This project will address elicitation of long-term human immune T and B cell memory to orthopox viruses following immunization of normal individuals and immunodeficient patients. Recent advances in understanding of mechanisms shunting TCR-based triggering away from activation-induced cell death have identified memory phenotype biomarkers. The murine TL MHC class Ib molecule, expressed during dendritic cell maturation, and its inducible CD8alpha,alpha homodimeric receptor on T cells serve as a "memory switch". The present proposal has three aims. First, as bioinformatic analysis of the human genome strongly suggests that HLA-F is the human orthologue of murine TL, we will express, refold and purify recombinant HLA-F, demonstrate its capacity to serve as a CD8alpha,alpha ligand and investigate receptor-ligand inducibility, using both monoclonal antibodies and HLA-F tetramers. The CD4 and CD8 subset distribution and stimuli for expression of CD8alpha,alpha and its ligand will be examined and linkage to memory phenotype established. Second, possible HLA-A2-restricted (and other) T cell epitopes shared by vaccinia, MVA and smallpox have been identified through genome-wide comparison in conjunction with position-specific scoring matrices, further parsed by proteosome cleavage site predictors. Those 1008 potential antigens will be synthesized, tested functionally using blood from vaccinated individuals by CFC assays, ELISPOT, proliferation and CTL assays as well as mass spectrometry. HLA-A2 tetramers containing the identified functional peptide epitopes will monitor elicitation of antigen-specific T cells, including induction, expansion and memory/effector phenotype development. Single cell-based PCR sequencing technology will detail the course of TCR usage during the immune response, ascertaining whether the antigen-specific repertoire becomes more focused or diverse and identify emergence of any HLA-A2-restricted public clonotypes. Once completed, other class I and class II MHC-restricted epitopes will be identified. Third, the nature of human neutralizing antibody responses elicited to orthopox immunization will be defined. Neutralization targets will be determined and the relative contribution of antibodies directed at individual viral proteins ascertained. Biophysical characterization of elicited human neutralizing monoclonal antibodies will be conducted. In this way, the rules regarding both the kinetics of B and T cell responses to their respective epitopes and concordance or discordance in immune targets will be defined.